The replication of influenza virus will be investigated. Studies in this laboratory indicate that the virus membrane (M) protein is found not only in plasma membranes but also in the nucleous and certain subcellular fractions of the infected cell. These findings will be uniquivocally confirmed by mapping the distribution of M. Distinction will be made between M and the non-structural (NS) protein and possible relationship between these two will be investigated. These aims will be carried out serologically and by biochemical methods which will separate and distinguish one protein from the other. Different cell lines and times of infection will be considered. Temperature-sensitive mutants which are available in this laboratory and which fail to synthesize M protein will facilitate this work considerably. The mechanism and sequence of assembly of virus proteins into the cell plasma membrane will be studied using temperature-sensitive mutants and wild-type virus of the WSN strain. Plasma membranes will be analyzed for protein patterns before and after infection to determine if displacement of cell protein occurs. Sequence of protein assembly will be carried out serologically by precipitating viral proteins from solubilized plasma membranes and by biological assay for activity. Continuation of studies using ts mutants should elucidate the role of the non-plaque-former in the infective process, the possible involvement of two RNA polymerases and the basis for recombination between virus particles defective in the synthesis of similar components.